PROJECT SUMMARY Multicellular life is defined by its organization of diverse cell types into higher-order tissues and organs. Millions of cells take part in a complex synthesis of gene expression, signaling cascades, and environmental sensing to maintain normal physiology. Disturbances in these processes can lead to illness like cancer, which results from aberrant cell growth and dysregulation. Understanding how cells behave at an individual level is therefore crucial to understanding both normal and abnormal physiological states. Previous investigations into cellular diversity have been limited by available technology. Bulk RNA sequencing (RNA-seq) averages transcriptomic data across all cells present in a sample; while this is sufficient for relatively homogeneous organs, it is inappropriate for complex tissues. Single-cell RNA-seq is now revealing previously unappreciated heterogeneity in systems long thought to have been uniform, such as stem cells, neutrophils, and skeletal muscle. In a typical experiment, tissues are dissociated into a single cell suspension; individual cells are isolated, either into a microwell or in a microfluidic droplet; cells are lysed; and mRNA is uniquely barcoded and collected. Throughput of these techniques ranges from a hundred to several thousands of transcriptomes obtained in a single workflow. As a result, investigators have discovered several new and rare cellular subtypes in heterogeneous tissues like the retina. While the ability to catalog cell types is rapidly increasing, the capacity to infer meaningful information about these cell types lags behind. Open problems include dissecting the gene regulatory networks governing cell states and deciding whether rare cell types are biologically meaningful. How do rare cell states arise and how are they developmentally related to common cell types? This proposal aims to develop a new technology, single cell calling cards, to address these concerns. The calling card method relies on fusing a transcription factor (TF) to a transposase which directs the insertion of a transposon into the genome near TF binding sites. These marks are recovered from genomic DNA and subsequently analyzed. Aim 1 of this proposal merges our existing method with single-cell RNA-seq to create single cell calling cards, which can simultaneously characterize cell identity and record TF binding sites. Calling card insertions are permanent and can be used to study TF localization throughout development. Moreover, they can serve as genealogical markers. Aim 2 develops new from single cell calling cards data to delineate phylogenetic relationships between cell types. Successful completion of these aims will yield new technologies to study how, and when, diverse cell fates arise.